A fluorescent probe derived from perylene compound and its preparation method and application

Abstract

The present invention discloses a fluorescent probe derived from a compound perylene and a preparation method and application thereof. The structural formula of the fluorescent probe derived from the compound perylene is shown in Formula 5. The present invention uses the compound perylene as a substrate molecule, and expands the conjugation range of the molecule by performing nitration, cyclization and other reaction modifications on the substrate molecule. The fluorescent probe molecule of the present invention can be used to label non-luminescent biomacromolecules, such as nucleic acid molecules, and detect changes in nucleic acid structure or properties through changes in fluorescence intensity. At the same time, the effective excitation of the fluorescent probe at an excitation wavelength of 405 nm allows it to be well utilized in some fixed excitation wavelength instruments, and the emitted blue fluorescence also broadens the selection range of researchers for fluorescent probes.

Description

Technical Field

[0001] The invention relates to a fluorescent probe derived from a perylene compound and a preparation method and application thereof. Background Art

[0002] Fluorescence labeling technology refers to the process of connecting a substance with fluorescence emission ability to a certain group of the molecule of the object being studied by covalent bonding or physical adsorption, and using fluorescence characteristics to reflect the molecular information of the object being studied. This technology has the advantages of no radioactive pollution, high sensitivity, and continuous real-time monitoring, and has been rapidly developed in the biomedical field. (Trac.Trends Anal.Chem.,2021,134,116143)

[0003] Nucleic acid is the genetic material of life and plays an important role in life activities. Nucleic acid molecule detection has important applications in disease diagnosis, environmental microbial identification, aptamers and other fields. Using fluorescent marker nucleic acid molecules for fluorescence detection is an important technology in molecular biology. Detecting changes in fluorescence intensity or wavelength helps to analyze the structure and property changes of nucleic acid molecules. (Proc. Natl. Acad. Sci. USA, 2005, 102, 17278-17283; J. Am. Chem. Soc. 2001, 123, 4928-4931) Although there are some endogenous fluorescent substances in living bodies, endogenous fluorescence alone cannot meet the current needs of molecular biology research. Therefore, synthesizing and screening more suitable fluorescent probes is a need for molecular biology research.

[0004] Perylene dyes are organic functional fluorescent dyes with large planar conjugated structures. They have the advantages of excellent photoelectric properties, high fluorescence quantum yield, and stable chemical properties. However, the large planar conjugated structure will reduce the water solubility of this type of fluorescent dye, limiting its application range. It is necessary to improve its water solubility by molecular modification and combining with highly soluble biological molecules or groups. (Chem. Eur. J., 2013, 19, 12846–12852) Summary of the invention

[0005] The present invention uses the compound perylene as a substrate molecule, and expands the conjugation range of the molecule by modifying the substrate molecule through reactions such as nitration and cyclization. In addition, it is connected to nucleotides by covalent bonds, thereby improving the fluorescence quantum efficiency and water solubility of the fluorescent probe molecule. Experimental results show that the fluorescent probe can emit blue fluorescence under 405nm excitation light. Therefore, for instruments with fixed excitation wavelengths (such as flow cytometers, etc.), on the one hand, the effective excitation of 405nm is useful; at the same time, the blue fluorescence emitted by the fluorescent probe also expands the researcher's selection range of blue fluorescent probes.

[0006] The technical solution of the present invention is as follows:

[0007] A perylene-derived fluorescent probe, the structural formula of which is shown in Formula 5:

[0008]

[0009] The synthesis method of the perylene-derived fluorescent probe of the present invention is as follows:

[0010] (1) Compound 1 (substrate perylene) undergoes a one-step nitration reaction with concentrated nitric acid (65%) under the catalysis of concentrated sulfuric acid (98%) to generate compound 2;

[0011] (2) Compound 2 is deoxygenated with triethyl phosphite to undergo a one-step Cadogan nitrogen heterocyclic reaction to obtain Compound 3;

[0012] (3) using a brominated ester to alkylate compound 3 to obtain compound 4;

[0013] (4) Compound 4 is hydrolyzed under alkaline conditions to obtain the target compound 5.

[0014] The synthetic route is as follows:

[0015]

[0016] Specifically, the operation method of step (1) is:

[0017] Dissolve compound 1 in 1,4-dioxane, then add concentrated sulfuric acid (catalytic amount) and concentrated nitric acid, react at 40-80° C. for 1-4 hours, and then post-treat to obtain compound 2;

[0018] The molar ratio of the compound 1 to nitric acid is 1:0.9 to 1:1.2;

[0019] The volume mass ratio (mL / g) of the 1,4-dioxane to the compound 1 is 70 to 125:1;

[0020] The post-treatment method is as follows: after the reaction is completed, the reaction solution is cooled to room temperature, poured into distilled water, a red solid is precipitated, and filtered with a Buchner funnel to obtain a crude product; the crude product is separated and purified by a chromatography column, with 200-300 mesh silica gel as the column filler, and a mixed solution of toluene / petroleum ether with a volume ratio of 1:3 as the eluent, the eluate containing the target compound is collected, and the solvent is removed by vacuum concentration to obtain compound 2.

[0021] Specifically, the operation method of step (2) is:

[0022] Compound 2 is mixed with triethyl phosphite, reacted at 120-200° C. for 5-12 hours, and then post-treated to obtain compound 3;

[0023] The volume mass ratio (mL / g) of triethyl phosphite to compound 2 is 10-20:1;

[0024] The post-treatment method is as follows: after the reaction is completed, the reaction solution is poured into petroleum ether and filtered to obtain a crude product; the crude product is separated and purified by a chromatography column, with 200-300 mesh silica gel as the column filler and a mixed solution of toluene / petroleum ether with a volume ratio of 1:1 as the eluent, the eluate containing the target compound is collected, and the solvent is removed by vacuum concentration to obtain compound 3.

[0025] Specifically, the operation method of step (3) is:

[0026] Compound 3 is dissolved in acetone, sodium hydride and bromoester are added, and the mixture is reacted at room temperature for 0.5 to 2 hours, followed by post-treatment to obtain compound 4;

[0027] The molar ratio of the compound 3 to sodium hydride and the brominated ester is 1:3:1.1 to 1:12:2;

[0028] The volume mass ratio (mL / g) of acetone to compound 3 is 100-500:1;

[0029] The post-treatment method is as follows: after the reaction is completed, quench with methanol, then pour the reaction solution into deionized water, extract with dichloromethane, collect the organic phase, dry with anhydrous sodium sulfate, and concentrate in vacuo to obtain a crude product; the crude product is separated and purified by a chromatography column, using neutral alumina as the column filler and a mixed solution of ethyl acetate and petroleum ether in a volume ratio of 1:1 as the eluent, collecting the eluate containing the target compound, and concentrating in vacuo to remove the solvent to obtain compound 4.

[0030] Specifically, the operation method of step (4) is:

[0031] Compound 4 is dissolved in N,N-dimethylformamide, and an aqueous NaOH solution is added, and the mixture is reacted at 30-60° C. for 1-4 hours, followed by post-treatment to obtain compound 5;

[0032] The volume mass ratio (mL / g) of the N,N-dimethylformamide to the compound 4 is 100 to 500:1;

[0033] The post-treatment method is as follows: after the reaction is completed, the reaction solution is cooled to room temperature, poured into deionized water, extracted with dichloromethane, the organic phase is collected, dried with anhydrous sodium sulfate, and concentrated in vacuo to obtain a crude product; the crude product is separated and purified by chromatography column, with 200-300 mesh silica gel as column filler, and a mixed solution of ethyl acetate / petroleum ether with a volume ratio of 1:2 as eluent, the eluate containing the target compound is collected, and the solvent is removed by vacuum concentration to obtain compound 5.

[0034] The perylene-derived fluorescent probe of the present invention can be used to label nucleic acid molecules. For example, compound 5 is coupled with NT-NH2 to obtain a nucleic acid molecule labeled with a fluorescent probe (abbreviated as NT-NP); the NT-NH2 is a nucleic acid molecule with an amino group modified at the end, and the nucleic acid sequence is any sequence.

[0035] The obtained fluorescent probe-labeled nucleic acid molecules (such as aptamers, etc.) have a wide range of applications (Applied Chemistry, 2018, 35, 28-39). For example:

[0036] Adenosine aptamer (APT-NP) labeled with fluorescent probe emits fluorescence under excitation. Graphene oxide (GO) is added (APT-NP+GO is obtained). The adsorption of graphene oxide on single-stranded nucleic acid can quench a certain amount of fluorescence. Finally, adenosine (ADENOSINE) is added (APT-NP+GO+ADENOSINE is obtained). The specific action of adenosine aptamer on adenosine causes the aptamer to desorb from graphene oxide, thereby restoring fluorescence (for experimental results, see Figure 1 ), so this assay can be used for the specific analysis of adenosine molecules.

[0037] The beneficial effects of the present invention are:

[0038] The perylene-derived fluorescent probe described in the present invention is a new fluorescent probe molecule, which can be used to label non-luminescent biomacromolecules, such as nucleic acid molecules, and detect changes in nucleic acid structure or properties through changes in fluorescence intensity. At the same time, the effective excitation of the fluorescent probe at an excitation wavelength of 405 nm enables it to be well utilized in some fixed excitation wavelength instruments, and the emitted blue fluorescence also broadens the range of researchers' choices for fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Fluorescently labeled adenosine aptamers were used for adenosine-specific analysis. DETAILED DESCRIPTION

[0040] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] Example 1 Synthesis of fluorescent probe compound 5

[0042] 1. Synthesis of compound 2:

[0043] Add 2g of compound 1 and 150mL of 1,4-dioxane to a 250mL three-necked flask, heat the reaction system to 80°C, and wait until compound 1 is completely dissolved. The system is a clear and transparent yellow solution. Cool the reaction system to 60°C, add 3 to 4 drops of concentrated sulfuric acid, and add 65% concentrated nitric acid drop by drop with a separatory funnel for a total of 4mL. The reaction system turns brick red, and the reaction progress is monitored by TLC. After the reaction is completed, after cooling to room temperature, pour the reaction solution into 400mL of distilled water, precipitate a large amount of red solid, and filter with a Buchner funnel to obtain a filter residue. The filter residue is separated and purified by a silica gel chromatography column (toluene / petroleum ether, V / V=1:3) to obtain 170mg of the target product compound 2, with a yield of 7.2%, and the properties are bright red crystals.

[0044] 2. Synthesis of compound 3:

[0045] 300 mg of compound 2 and 5 mL of triethyl phosphite were added to a 50 mL three-necked flask, and the reaction mixture was reacted at 130 ° C for 8 h, and then cooled to room temperature. The reaction mixture was poured into 100 mL of petroleum ether solution and filtered to obtain a crude product. The crude product was separated and purified by a silica gel chromatography column (toluene / petroleum ether, V / V=1:1) to obtain 220 mg of the target product compound 3, with a yield of 82%, and the properties were orange crystals.

[0046] 3. Synthesis of compound 4:

[0047] Add 100mg of compound 3 and 20mL of acetone to a 100mL three-necked flask, stir until the reactant is fully dissolved, add 0.4g of sodium hydride in batches, and then add 0.3g of 1-bromobutyric acid ethyl ester dropwise. After stirring at room temperature for 1h, add 1mL of methanol to quench the reaction mixture. Then pour the reaction mixture into 20mL of deionized water and extract with dichloromethane, using 40mL of dichloromethane each time, for a total of three extractions. Collect the organic phase, dry it with anhydrous sodium sulfate, and obtain a solid under vacuum concentration. The solid is further separated and purified by a neutral alumina chromatography column (ethyl acetate / petroleum ether, V / V=1:1) to obtain 110mg of the target product compound 4-a, with a yield of 77%, and the properties are yellow-green solid.

[0048] Structural analysis by NMR spectroscopy: 1H NMR (400MHz, CDCl3) δ8.64(d,J=7.5Hz,2H),8.12(d,J=8.0Hz,2H),7.91(d,J=8.8Hz,2H),7.87–7.72(m,4H),4. 78(t,J=6.7Hz,2H), 4.10(q,J=7.1Hz,2H), 2.42(p,J=6.8Hz,2H), 2.30(t,J=7.1Hz,2H), 1.20(t,J=7.1Hz,3H).

[0049] By changing the chain length of the ester, 1-bromobutyric acid ethyl ester was replaced by 1-bromoethyl ester and 1-bromodecanoic acid ethyl ester, respectively, to synthesize compound 4-b and compound 4-c:

[0050] Add 100mg of compound 3 and 20mL of acetone to a 100mL three-necked flask, stir until the reactant is fully dissolved, add 0.4g of sodium hydride in batches, and then add 0.3g of ethyl 1-bromoacetate dropwise. After stirring at room temperature for 1h, add 1mL of methanol to quench the reaction mixture. Then pour the reaction mixture into 20mL of deionized water and extract with dichloromethane, using 40mL of dichloromethane each time, for a total of three extractions. Collect the organic phase, dry it with anhydrous sodium sulfate, and obtain a solid under vacuum concentration. The solid is further separated and purified by a neutral alumina chromatography column (ethyl acetate / petroleum ether, V / V=1:1) to obtain 110mg of the target product compound 4-b, with a yield of 77%, and the properties are yellow-green solid.

[0051] Structural analysis by NMR spectroscopy: 1 H NMR (400MHz, CDCl3) δ8.64(d,J=7.5Hz,2H),8.12(d,J=8.0Hz,2H),7.91(d,J=8.8Hz,2H) ,7.87–7.72(m,4H),4.81(t,J=6.7Hz,2H),4.16(q,J=7.1Hz,2H),1.21(t,J=7.1Hz,3H).

[0052] Add 100mg of compound 3 and 20mL of acetone to a 100mL three-necked flask, stir until the reactant is fully dissolved, add 0.4g of sodium hydride in batches, and then add 0.3g of ethyl 1-bromodecanoate dropwise. After stirring at room temperature for 1h, add 1mL of methanol to quench the reaction mixture. Then pour the reaction mixture into 20mL of deionized water and extract with dichloromethane, using 40mL of dichloromethane each time, for a total of three extractions. Collect the organic phase, dry it with anhydrous sodium sulfate, and obtain a solid under vacuum concentration. The solid is further separated and purified by a neutral alumina chromatography column (ethyl acetate / petroleum ether, V / V=1:1) to obtain 110mg of the target product compound 4-c, with a yield of 77%, and the properties are yellow-green solid.

[0053] Structural analysis by NMR spectroscopy: 1 H NMR (400MHz, CDCl3) δ8.64(d,J=7.5Hz,2H),8.12(d,J=8.0Hz,2H),7.91(d,J=8.8Hz,2H),7.87–7.72(m,4H),4.81(t,J=6.7Hz,2 H), 4.16 (q, J = 7.1Hz, 2H), 2.32 (t, J = 7.1Hz, 2H), 1.74 (m, 2H), 1.66 (dd, 2H), 1.26–1.33 (m, J = 6.8Hz, 10H), 1.13 (t, J = 7.1Hz, 3H).

[0054] 4. Synthesis of compound 5:

[0055] Add 100mg of compound 4-a and 20mL of N,N-dimethylformamide to a 50mL three-necked flask, stir until the reactant is fully dissolved, then add 5mL of 2mol / L NaOH aqueous solution, heat to 40℃ and stir for 3h. After the reaction mixture is cooled to room temperature, pour it into 60mL of deionized water and extract with dichloromethane, using 40mL of dichloromethane each time, for a total of three extractions. Collect the organic phase, dry it with anhydrous sodium sulfate, and obtain a solid under vacuum concentration. The solid is further separated and purified by silica gel chromatography column (ethyl acetate / petroleum ether, V / V=1:2) to obtain 70mg of the target product compound 5-a, with a yield of 76%, and the properties are yellow-green solid.

[0056] Structural analysis by NMR spectroscopy: 1 H NMR(400MHz,DMSO)δ12.19(s,1H),8.77(d,J=7.5Hz,2H),8.20(d,J=8.0Hz,2H),8 .12–7.91(m,4H),7.83(t,J=7.8Hz,2H),4.86(t,J=6.2Hz,2H),2.39–2.17(m,4H).

[0057] By replacing compound 4-a with compound 4-b, compound 5-b is obtained. 1 H NMR (400MHz, DMSO) δ12.19(s,1H),8.77(d,J=7.5Hz,2H),8.20(d,J=8.0Hz,2H),8.12–7.91(m,4H),7.83(t,J=7.8Hz,2H),4.91(s,2H).

[0058] By replacing compound 4-a with compound 4-c, compound 5-c is obtained. 1 H NMR (400MHz, DMSO) δ12.19(s,1H),8.77(d,J=7.5Hz,2H),8.20(d,J=8.0Hz,2H),8.12–7.91(m,4H),7.83( t,J=7.8Hz,2H),4.86(t,J=6.2Hz,2H),2.97(t,J=6.2Hz,2H),2.39(t,J=6.1Hz,2H),1.96–2.13(m,12H).

[0059] Example 2 Fluorescent probe labeling of nucleic acid molecules

[0060] 1. Activation of NT-NH2 molecules

[0061] NT-NH2 was prepared into a 100 μM solution using 10 mM Tris buffer solution (pH=8.0), which was placed in a thermal cycler. The temperature was rapidly raised to 95°C for 10 min according to the set program, and then slowly lowered to 37°C to obtain a NT-NH2 buffer solution for use.

[0062] 2. Coupling reaction of NT-NH2 molecules

[0063] Weigh 1 mg of compound 5-a and dissolve it in N,N-dimethylformamide (DMF), add 10 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) aqueous solution respectively, so that compound 5:EDC:NHS (molar ratio) = 1:2:2, react and activate for 1 h under light-proof conditions, and generate compound 6 in the system; then add the reaction mixture to the NT-NH2 buffer solution reserved in the previous step, seal and react for 4 h under shaking conditions and protect from light.

[0064] 3. Purification of fluorescent probe-coupled nucleic acid molecules (abbreviated as: NT-NP)

[0065] (1) Ethanol precipitation

[0066] In the system where the reaction in step 2 is completed, anhydrous ethanol (reaction system / anhydrous ethanol, V / V=1:3) is added, and then placed in a -4°C environment overnight to precipitate the nucleic acid molecules. After taking it out of the refrigerator, put it directly into a refrigerated centrifuge, centrifuge it at -4°C, 12000rpm for 20min, and discard the supernatant. Then add 75% ethanol, blow and wash, put it into the centrifuge again, centrifuge it at -4°C, 12000rpm for 20min, discard the supernatant, repeat the above steps three times, and obtain the crude product of NT-NP.

[0067] (2) NAP-5 desalting

[0068] The obtained crude product was dissolved in double distilled water, desalted using a NAP-5 desalting column to obtain 500 μL of NT-NP solution, and precipitated using the ethanol precipitation method described in the previous step to obtain desalted fluorescent probe-labeled nucleic acid molecules.

[0069] The reaction formula is as follows:

[0070]

[0071] Compounds 5-b and 5-c with different side chain lengths can also be used to label nucleic acid molecules according to the above method.

Claims

1. A perylene-derived fluorescent probe, the structural formula of which is shown in Formula 5:

2. The method for synthesizing the perylene-derived fluorescent probe according to claim 1, wherein: The synthetic method is: (1) Compound 1 undergoes a one-step nitration reaction with concentrated nitric acid under the catalysis of concentrated sulfuric acid to generate compound 2; (2) Compound 2 is deoxygenated with triethyl phosphite to undergo a one-step Cadogan nitrogen heterocyclic reaction to obtain Compound 3; (3) using a brominated ester to alkylate compound 3 to obtain compound 4; (4) Compound 4 is hydrolyzed under alkaline conditions to obtain the target compound 5; The synthetic route is as follows:

3. The synthesis method according to claim 2, characterized in that The operation method of step (1) is: Dissolve compound 1 in 1,4-dioxane, then add concentrated sulfuric acid and concentrated nitric acid, react at 40-80° C. for 1-4 hours, and then post-treat to obtain compound 2; The molar ratio of the compound 1 to nitric acid is 1:0.9 to 1:1.

2.

4. The synthesis method according to claim 2, characterized in that The operation method of step (2) is: Compound 2 is mixed with triethyl phosphite, reacted at 120-200° C. for 5-12 hours, and then post-treated to obtain compound 3; The volume mass ratio of the triethyl phosphite to the compound 2 is 10 to 20:

1.

5. The synthesis method according to claim 2, characterized in that The operation method of step (3) is: Compound 3 is dissolved in acetone, sodium hydride and bromoester are added, and the mixture is reacted at room temperature for 0.5 to 2 hours, followed by post-treatment to obtain compound 4; The molar ratio of the compound 3 to sodium hydride and the brominated ester is 1:3:1.1 to 1:12:

2.

6. The synthesis method according to claim 2, characterized in that The operation method of step (4) is: Compound 4 was dissolved in N,N-dimethylformamide, and an aqueous NaOH solution was added, and the mixture was reacted at 30-60° C. for 1-4 h, followed by post-treatment to obtain compound 5.

7. A fluorescent probe-labeled nucleic acid molecule obtained by coupling a fluorescent probe derived from the compound of formula 5 in claim 1 with NT-NH2; the NT-NH2 is a nucleic acid molecule with a terminal modified amino group, and the nucleic acid sequence is an arbitrary sequence.

8. Use of the nucleic acid molecule labeled with the fluorescent probe as claimed in claim 7 in the specific analysis of adenosine molecules.

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